RNA viruses have very high mutation frequencies. When a RNA virus replicates, nucleotide mutations are generated resulting in a population of variants. The consensus sequence, which is used to define a RNA virus, represents the genetic average of every nucleotide position along the genome. The population of RNA virus variants is a network of variants organized in sequence space around the consensus sequence. This mutant spectrum is often referred to as quasispecies.
This genetic diversity creates a cloud of mutations that are potentially beneficial to viral survival, whereby creating an antigenic drift that requires frequent updates of vaccines and providing the basis for resistance to antivirals. It is known that altering the ability of a RNA virus to generate a normal mutation frequency, reduces viral fitness (i.e., the relative ability of a given virus to generate progeny viruses, taking into account all aspects of the virus life cycle including replication) and attenuates the virus during in vivo infection.
Reducing the fitness of RNA viruses may also be achieved by affecting replication or translation, through a variety of means, including altering codon pair bias.
Another feature that may affect RNA virus fitness is mutational robustness and/or sequence space. Mutational robustness is the ability to conserve phenotype in light of genetic changes (neutral mutation). However, little is known about the effects induced by alteration of RNA virus mutational robustness. Some studies addressed the indirect alteration of RNA virus mutational robustness, using constructs designed to alter fitness by other mechanisms, such as codon deoptimization (e.g., alteration of codon bias and codon pair bias). Therefore, these studies did not address mutational robustness per se (Lauring et al. 2012; Coleman et al. 2008).
The attenuation of RNA viruses for vaccine production faces the problem of genetic instability and of the associated risk of genetic reversion or mutation to a pathogenic phenotype.
The conventional method for RNA virus attenuation currently involves the introduction of random gene mutation or passages in unnatural conditions, whereby introducing more mutations than those actually required for attenuation, but lowering the risk of genetic reversion. This step is mostly empirical and is rather specific of the particular RNA virus type or species under attenuation.
Hence, the current method for RNA virus attenuation involves events, which depend on chance and cannot be universally applied to a variety of virus types.
The application provides means for RNA virus attenuation, which are non-empirical and which can be applied to all RNA viruses.
The means of the application are rationally based on the alteration of mutational robustness and/or of the localization of the virus in sequence space.